Soybean - Patents granted to and patent applications filed by Pioneer Hi-Bred

A method for transforming soybean cells, comprising the steps of

(A) providing a complete plant medium that supports rapid division of plant cells, said medium comprising
(i) a virulence-inducing amount of a signal molecule,
(ii) a growth promoting amount of an auxin, and
(iii) Agrobacterium tumefaciens bacteria in log growth phase, such that said bacteria are present in said medium in a concentration of about 108 viable cells per ml, wherein said bacteria contain a chimeric gene and said medium is buffered at a pH below 6.0;
(B) introducing into a first portion of said medium a plurality of germinated soybean seeds, from each of which seeds seed coat and radicle have been removed, and separating the cotyledons of each seed so as to expose the cotyledonary node of each seed, whereby a plurality of explants is produced; then
(C) macerating said cotyledonary node, without cutting entirely through each of said explants to the abaxial side thereof, and thereafter maintaining said explants in said first portion, at room temperature, for at least about 30 minutes; then
(D) transferring said explants to a second portion of said medium in solidified form, such that said explants are embedded in said medium, adaxial side up and level with the surface of said medium, and culturing said explants for about 3 days at about 22° C.;
(E) treating said explants in counterselection medium;
(F) cultivating said explants in agarose-solidified selection medium, wherein said explants are embedded adaxial side down in said selection medium, whereby transformed cells in said explants are favored; and then
(G) selecting transformed cells from said explants.

A genotype-independent method for producing a transgenic soybean plant which comprises:

A) co-cultivating an explant derived from a hypocotyl or cultured cotyledonary nodes of a germinated soybean seed with cells of an Agrobacterium species containing a chimeric gene at a concentration of 10⁸ to 3 x 10⁸ cells/ml and in the presence of a signal compound selected from the group consisting of acetosyringone, alfa-hydroxyacetosyringone, acetovanillone, syringaldehyde, syringic acid, sinapinic acid and mixtures thereof;
B) maintaining a temperature for co-cultivation of from 18 to 28°C; and
C) inducing virulence of the Agrobacterium by decreasing the pH of the plant culture media below pH 6.0.

A genotype-independent method for producing a transgenic soybean plant which comprises:

A) co-cultivating an explant derived from a hypocotyl or cultured cotyledonary node of a germinated soybean seed with cells of an Agrobacterium species containing a chimeric gene at a concentration of 10⁸ to 3 x 10⁸ cells/ml and in the presence of a signal compound selected from the group consisting of acetosyringone, alpha-hydroxyacetosyringone, acetovanillone, syringaldehyde, syringic acid, sinapinic acid and mixtures thereof;
B) maintaining a temperature for co-cultivation of from about 22 to about 25°C;
C) inducing virulence of the Agrobacterium by decreasing the pH of the plant culture media below pH 6.0; and
D) regenerating a transgenic soybean plant.

A genotype-independent method for producing a transgenic soybean plant which comprises:

a. cocultivating an explant derived from a hypocotyl or cultured cotyledonary nodes of a germinated soybean seed with Agrobacterium species containing a chimeric gene in the presence of a signal compound selected from the group consisting of acetosyringone, a-hydroxyacetosyringone, acetovanillone, syringaldehyde, syringic acid, and sinapinic acid and mixtures thereof;
b. maintaining a temperature for cocultivation of from 18 to 28°C; and
c. inducing virulence of the Agrobacterium by decreasing the pH of the plant culture media below pH 6.0.